三七总皂苷对缺氧复氧致皮质神经元损伤细胞凋亡相关基因及蛋白表达的影响

Effect of PNS on expression of apoptosis-relating mRNA and protein in impaired CNC caused by hypoxia/re-oxygenation

[目的]研究三七总皂苷(PNS)对缺氧复氧致皮质神经元损伤细胞凋亡相关基因及蛋白表达的影响,探讨PNS抗神经元缺氧/复氧诱导的细胞凋亡的作用。[方法]体外原代培养大鼠胎鼠大脑皮质神经元细胞(CNC),采用缺氧/复氧(H/R)诱导皮质神经元细胞氧化应激损伤模型,采用PNS50、10、2mg/L进行干预。采用实时荧光定量逆转录酶-聚合酶链式反应(RT-PCR)技术检测B细胞淋巴瘤-2(Bcl-2)、B细胞淋巴瘤-2相关蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)mRNA表达的变化,采用福林-酚法(Lowrry)法检测caspase-3蛋白含量的变化。探讨PNS抑制神经元细胞凋亡的分子机制。[结果]PNS50、10mg/L剂量组可以显著降低Bax表达;PNS50mg/L组可以显著增加Bcl-2表达;PNS有降低caspase-3表达的趋势。PNS各剂量组均能够抑制cas-pase-3的活性,有剂量依赖性趋势。[结论]PNS可能是通过增加抑制凋亡基因Bcl-2的表达,抑制促凋亡基因Bax的表达,抑制caspase-3的活性等作用抑制神经元缺氧/复氧诱导的细胞凋亡。

[Objective] In order to research the protective effect of PNS against apoptosis, we observed the effects of PNS on the mRNA expression of Bcl-2, Bax, caspase-3 and caspase-3 activity in impaired CNC model caused by hypoxia/re-oxygenation. [Methods] We cultured cortical neuronal cell （CNC） of rat in vitro and established an impaired cell model by hypoxia/re-oxygenation （H/R）. We treated the impaired cells with three dosages, 50 mg/L, 10 mg/L, 2 mg/L of PNS, and studied the mRNA expression of Bcl-2, Bax, caspase-3 by the method of real time RT-PCR. We studied the protein activity of caspase-3 by the method of Lowrry. [Results] 50 mg/L, 10 mg/L of PNS decreased the expression of Bax signifi- cantly compared with H/R group. 50 mg/L of PNS increased the expression of Bcl-2 significantly compared with H/R group. H/R group had a tendency of up-regulating and PNS group had a tendency of down-regulating the expression of caspase-3. These three dosages of PNS inhibited the protein activity of caspase-3 with a tendency of dose -dependent manner. [Conclusion] The possible protective mechanism of PNS against apoptosis may be by decreasing the expression of Bax, increasing the expression of Bel-2 and inhibiting the protein activity of caspase-3.